Pneumococcal sialidase promotes bacterial survival by fine-tuning of pneumolysin-mediated membrane disruption.

Pneumolysin (Ply) is an indispensable cholesterol-dependent cytolysin for pneumococcal infection. Although Ply-induced disruption of pneumococci-containing endosomal vesicles is a prerequisite for the evasion of endolysosomal bacterial clearance, its potent activity can be a double-edged sword, having a detrimental effect on bacterial survivability by inducing severe endosomal disruption, bactericidal autophagy, and scaffold epithelial cell death. Thus, Ply activity must be maintained at optimal levels. We develop a highly sensitive assay to monitor endosomal disruption using NanoBiT-Nanobody, which shows that the pneumococcal sialidase NanA can fine-tune Ply activity by trimming sialic acid from cell-membrane-bound glycans. In addition, oseltamivir, an influenza A virus sialidase inhibitor, promotes Ply-induced endosomal disruption and cytotoxicity by inhibiting NanA activity in vitro and greater tissue damage and bacterial clearance in vivo. Our findings provide a foundation for innovative therapeutic strategies for severe pneumococcal infections by exploiting the duality of Ply activity.

Prime-boost-type PspA3 + 2 mucosal vaccine protects cynomolgus macaques from intratracheal challenge with pneumococci.

BACKGROUND: Although vaccination is recommended for protection against invasive pneumococcal disease, the frequency of pneumococcal pneumonia is still high worldwide. In fact, no vaccines are effective for all pneumococcal serotypes. Fusion pneumococcal surface protein A (PspA) has been shown to induce a broad range of cross-reactivity with clinical isolates and afford cross-protection against pneumococcal challenge in mice. Furthermore, we developed prime-boost-type mucosal vaccines that induce both antigen-specific IgG in serum and antigen-specific IgA in targeted mucosal organs in previous studies. We investigated whether our prime-boost-type immunization with a fusion PspA was effective against pneumococcal infection in mice and cynomolgus macaques. METHODS: C57BL/6 mice were intramuscularly injected with fusion PspA combined with CpG oligodeoxynucleotides and/or curdlan. Six weeks later, PspA was administered intranasally. Blood and bronchoalveolar lavage fluid were collected and antigen-specific IgG and IgA titers were measured. Some mice were given intranasal Streptococcus pneumoniae and the severity of infection was analyzed. Macaques were intramuscularly injected with fusion PspA combined with CpG oligodeoxynucleotides and/or curdlan at week 0 and week 4. Then, 13 or 41 weeks later, PspA was administered intratracheally. Blood and bronchoalveolar lavage fluid were collected and antigen-specific IgG and IgA titers were measured. Some macaques were intranasally administered S. pneumoniae and analyzed for the severity of pneumonia. RESULTS: Serum samples from mice and macaques injected with antigens in combination with CpG oligodeoxynucleotides and/or curdlan contained antigen-specific IgG. Bronchial samples contained antigen-specific IgA after the fusion PspA boosting. This immunization regimen effectively prevented S. pneumoniae infection. CONCLUSIONS: Prime-boost-type immunization with a fusion PspA prevented S. pneumoniae infection in mice and macaques.

Establishment of a modified opsonophagocytic killing assay for anti-pneumococcal surface protein A antibody.

Streptococcus pneumoniae (pneumococcus) is a pathogenic gram-positive bacterium that causes pneumonia, meningitis, and sepsis. Pneumococcal surface protein A (PspA) induces antibodies that protect against lethal infections by pneumococci. PspA is a choline-binding protein present on the cell surface of almost all pneumococcal strains and is a non-capsular polysaccharide vaccine candidate. For research and development of PspA-based vaccines, an in-vitro test system to measure the activity of functional antibodies capable of killing pneumococci is essential. The opsonophagocytic killing (OPK) assay is used to evaluate the opsonic activity of functional antibodies induced by capsular polysaccharide (CPS)-based vaccines (standard OPK assay). Despite the potential of anti-PspA antibodies to protect against lethal infections in mice, the standard OPK assay fails to evaluate anti-PspA antibodies. Using a pneumococcal surface protein C-deficient strain and extending the incubation time of opsonized bacteria, complement, and HL-60 cells reportedly results in enhanced bactericidal activity (modified OPK assay). We aimed to measure the bactericidal activity of anti-PspA antibodies in intact pneumococcal strains. We optimized the pneumococcal culture method used in the OPK assay to increase the efficiency of anti-PspA antibody-mediated phagocytosis of HL-60 cells. As thick capsules hinder phagocytosis, we attempted to obtain pneumococci with thin capsules through an improved culture method. As pneumococci attached to cells exhibit thin capsules, pneumococci cultured in Todd Hewitt yeast extract (THY) broth were spread on blood agar plates and incubated for 4 h. cpsA mRNA transcript levels in pneumococci cultured on blood agar were lower than those in pneumococci cultured in THY broth. OPK activity against pneumococci expressing PspA of clades 1-5 was reasonably well detected using pneumococci cultured on blood agar in the modified OPK assay. The modified OPK assay for anti-PspA antibody using pneumococci cultured on blood agar represents a useful assay to determine the killing activity of functional anti-PspA antibodies against pneumococci.

A bacterial small RNA regulates the adaptation of Helicobacter pylori to the host environment.

Long-term infection of the stomach with Helicobacter pylori can cause gastric cancer. However, the mechanisms by which the bacteria adapt to the stomach environment are poorly understood. Here, we show that a small non-coding RNA of H. pylori (HPnc4160, also known as IsoB or NikS) regulates the pathogen's adaptation to the host environment as well as bacterial oncoprotein production. In a rodent model of H. pylori infection, the genomes of bacteria isolated from the stomach possess an increased number of T-repeats upstream of the HPnc4160-coding region, and this leads to reduced HPnc4160 expression. We use RNA-seq and iTRAQ analyses to identify eight targets of HPnc4160, including genes encoding outer membrane proteins and oncoprotein CagA. Mutant strains with HPnc4160 deficiency display increased colonization ability of the mouse stomach, in comparison with the wild-type strain. Furthermore, HPnc4160 expression is lower in clinical isolates from gastric cancer patients than in isolates derived from non-cancer patients, while the expression of HPnc4160's targets is higher in the isolates from gastric cancer patients. Therefore, the small RNA HPnc4160 regulates H. pylori adaptation to the host environment and, potentially, gastric carcinogenesis.

Bacteria-Induced Group 2 Innate Lymphoid Cells in the Stomach Provide Immune Protection through Induction of IgA.

The intestinal microbiota shapes and directs immune development locally and systemically, but little is known about whether commensal microbes in the stomach can impact their immunological microenvironment. Here, we report that group 2 innate lymphoid cells (ILC2s) were the predominant ILC subset in the stomach and show that their homeostasis and effector functions were regulated by local commensal communities. Microbes elicited interleukin-7 (IL-7) and IL-33 production in the stomach, which in turn triggered the propagation and activation of ILC2. Stomach ILC2s were also rapidly induced following infection with Helicobacter pylori. ILC2-derived IL-5 resulted in the production of IgA, which coated stomach bacteria in both specific pathogen-free (SPF) and H. pylori-infected mice. Our study thus identifies ILC2-dependent IgA response that is regulated by the commensal microbiota, which is implicated in stomach protection by eliminating IgA-coated bacteria including pathogenic H. pylori.

Mutational diversity in mutY deficient Helicobacter pylori and its effect on adaptation to the gastric environment.

Helicobacter pylori, a pathogenic bacterium that colonizes in the human stomach, harbors DNA repair genes to counter the gastric environment during chronic infection. In addition, H. pylori adapts to the host environment by undergoing antigenic phase variation caused by genomic mutations. The emergence of mutations in nucleotide sequences is one of the major factors underlying drug resistance and genetic diversity in bacteria. However, it is not clear how DNA repair genes contribute to driving the genetic change of H. pylori during chronic infection. To elucidate the physiological roles of DNA repair genes, we generated DNA repair-deficient strains of H. pylori (ΔuvrA, ΔuvrB, ΔruvA, Δnth, ΔmutY, ΔmutS, and Δung). We performed susceptibility testing to rifampicin in vitro and found that ΔmutY exhibited the highest mutation frequency among the mutants. The number of bacteria colonizing the stomach was significantly lower with ΔmutY strain compared with wild-type strains in a Mongolian gerbil model of H. pylori infection. Furthermore, we performed a genomic sequence analysis of the strains isolated from the Mongolian gerbil stomachs eight weeks after infection. We found that the isolated ΔmutY strains exhibited a high frequency of spontaneous G:C to T:A mutations. However, the frequency of phase variations in the ΔmutY strain was almost similar to the wild-type strain. These results suggest that MutY may play a role in modes of gastric environmental adaptation distinct from phase variation.

A new microsporidium Percutemincola moriokae gen. nov., sp. nov. from Oscheius tipulae: A novel model of microsporidia-nematode associations.

Here, we describe a new microsporidium Percutemincola moriokae gen. nov., sp. nov., which was discovered in the intestinal and hypodermal cells of a wild strain of the nematode Oscheius tipulae that inhabits in the soil of Morioka, Iwate Prefecture, Japan. The spores of Pe. moriokae had an average size of 1.0 × 3.8 µm and 1.3 × 3.2 µm in the intestine and hypodermis, respectively, and electron microscopy revealed that they exhibited distinguishing features with morphological diversity in the hypodermis. Isolated spores were able to infect a reference strain of O. tipulae (CEW1) through horizontal transmission but not the nematode Caenorhabditis elegans. Upon infection, the spores were first observed in the hypodermis and then in the intestine the following day, suggesting a unique infectious route among nematode-infective microsporidia. Molecular phylogenetic analysis grouped this new species with the recently identified nematode-infective parasites Enteropsectra and Pancytospora forming a monophyletic sister clade to Orthosomella in clade IV, which also includes human pathogens such as Enterocytozoon and Vittaforma. We believe that this newly discovered species and its host could have application as a new model in microsporidia-nematode association studies.